PROJECT SUMMARY/ ABSTRACT Background: Long QT Syndrome Type 2 (LQT2) is characterized by mutations in the cardiac K+ channel known as hERG (human ether-a-go-go related gene) channel or Kv11.1, encoded by the KCNH2 gene. Mutations in the hERG channel result in a decrease in an important cardiac, repolarizing current: the rapidly activating delayed rectifier K+ current (IKr). The fundamental defect in LQTS is prolonged ventricular repolarization, caused by the imbalance of inward and outward currents, and an increase in occurrences of life-threatening arrhythmias? such as torsades de pointes polymorphic ventricular tachycardia, which can lead to ventricular fibrillation and sudden cardiac death. LQTS manifests not only from mutations in cardiac ion channels (as in LQT1, LQT2, and LQT3), but also mutations in ion channel interacting proteins (such as calmodulin in LQT14-16). A recent study revealed RNF207 as a potential modifier of the hERG channel. The hypothesis is that RNF207 alters cardiac repolarization through its interaction with the hERG K+ channel. Methods and Objectives: This interaction will be examined by looking at wild-type and mutant variants of hERG and RNF207. Mutant variants of interest will be hERG T613M and RNF207 G603fs. Feasibility for this project has been verified by positive co-localization near cardiac t-tubules seen in preliminary immunofluorescent experiments and multi- protein complex formation in co-immunoprecipitation experiments. The extent and nature of this interaction will be determined using a combination of biochemistry and electrophysiology techniques. Aim 1 will verify the protein-protein interaction using immunofluorescence (visualized with both confocal and standard emission depletion microscopy), co-immunoprecipitation, and GST pull-down assays. Aim 2 will analyze the functional interaction between hERG and RNF207 in human induced pluripotent stem cell- derived cardiomyocytes (hiPSC- CMs) and HEK 293 cells using whole-cell patch-clamp techniques. Conclusion: Our study will further reveal the cellular and molecular effects of RNF207 on hERG channels and ventricular repolarization. Furthermore, we will test the mechanisms for the underlying hypothesis that polygenic variability influences LQTS severity. The value of the proposed research lies in its ability to provide insights into novel mechanisms for LQTS and elucidate possible targets for new therapeutic treatments.